Anti-depression pharmaceutical composition containing Polygala extract

ABSTRACT

A pharmaceutical composition contains a therapeutically effective amount of an active ingredient, in admixture with a pharmaceutically acceptable carrier or diluent for the active ingredient, in which the active ingredient is i) a polar solvent extract of  Polygala , the polar solvent being water or a mixture of water and methanol or ethanol; ii) an aqueous fraction resulting from an extraction of the polar solvent extract with an organic solvent; iii) an organic eluate by introducing the polar solvent extract or the aqueous fraction into a reverse phase chromatography column, and eluting the column with water and an organic solvent; or iv) a filtrate having a molecular mass less than 30000 in the organic eluate.

This application is a divisional of application Ser. No. 10/335,920,filed Jan. 3, 2003 now U.S. Pat. No. 6,911,222 (of which the entiredisclosure is hereby incorporated by reference); which is a divisionalof application Ser. No. 10/103,288, filed Mar. 22, 2002, now abandoned.

FIELD OF THE INVENTION

The present invention is related to a method of preparing apharmaceutical composition from Polygala, and in particular to ananti-depression pharmaceutical composition containing polygala extract.

BACKGROUND OF THE INVENTION

P. tenuifolia Willd. And P. sibirica L. are traditional Chinese herbmedicines. Generally the root as a whole or only cortex of the root ofwhich are taken as a medicine useful in tranquilizing a patient,treating coughing and releasing inflammation. It was reported by Koo etal. that an aqueous extract of polygala tenuifolia WILLDENOW(polygalaceae) root may prevent the ethanol-induced cytoxicity in Hep G2cells through inhibitition of the apoptosis of Hep G2 cells [Koo H N,Jeong H J, Kim K R, Kim J C, Kim K S, Kang B K, Kim H M and Kim J J.Immunopharmacology & Immunotoxicology. 22(3): 531–44, 2000]. Kim et al.also reported that an aqueous extract of polygala tenuifolia root mayinhibit tumor necrosis factor-alpha secretion by inhibitinginterleukin-1 secretion, and has an anti-inflammatory activity on thecentral nervous system [Kim H M, Lee E H, Na H J, Lee S B, Shin T Y, LyuY S, Kim N S and Nomura S. Journal of Ethnopharmacology. 61(3): 201–8,1998]. Furthermore, it was reported that euxanthone may be one of theneuropharmacological active compounds in the medicinal plant polygalaaudata [Mak N K, Li W K, Zhang M, Wong R N, Tai L S, Yung K K and LeungH W. Life Sciences. 66(4): 347–54, 2000]. The saponins of polygala werereported having the potential to be used as vaccine adjuvants toincrease specific immune responses [Estrada A, Katselis G S, Laarveld Band Barl B. Comparative Immunology, Microbiology & Infectious Diseases.23(1): 27–43, 2000] and having significant immunological properties[Desbene S, Hanquet B, Shoyama Y, Wagner H and Lacaille-Dubois M A.Journal of Natural Products. 62(6): 923–6, 1999]. Other pharmacologicalactivities can also be found in the literature, but none of themsuggests an anti-depression activity.

SUMMARY OF THE INVENTION

A primary objective of the present invention is to provide a method ofpreparing a pharmaceutical composition from Polygala, and in particularto an anti-depression pharmaceutical composition.

In the present invention extracts prepared from the dry whole root orthe cortex of the root of Polygala by using various solvents forexamples water, ethanol, ethyl acetate and hexane were investigated asto the anti-depression activity thereof, and polar extracts preparedfrom water or a mixture of water and ethanol or methanol were foundsignificantly potent among them. Further separations/partitions of thepolar extracts were carried out in the present invention and theiranti-depression activity was evaluated, so that fractions havingsignificant potency in anti-depression activity are screened. Further,the present invention also discloses a method to prepare a compositioncontaining pharmaceutically potent components, and it is non-toxic.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses an anti-depression pharmaceuticalcomposition comprising a therapeutically effective amount of an activeingredient, in admixture with a pharmaceutically acceptable carrier ordiluent for the active ingredient, in which the active ingredient is i)a polar solvent extract of Polygala, the polar solvent being water or amixture of water and methanol or ethanol; ii) an aqueous fractionresulting from an extraction of the polar solvent extract with anorganic solvent; iii) an organic eluate by introducing the polar solventextract or the aqueous fraction into a reverse phase chromatographycolumn, and eluting the column with water and an organic solvent; or iv)a filtrate having a molecular mass less than 30000 Dalton in the organiceluate.

The Polygala which can be used in the invention includes (but notlimited to) P. tenuifolia Willd., P. sibirica L., P. sibirica var.megalopha Franch., P. japonica Houtt., P. hybrida DC, P. arillataBuch.-Ham ex D. Don, P. glomerata Lour., P. arvensis Willd., P.tatarinowii, P. fallax Hemsl., P. wattersii Hance, P. hongkongensis varstenophylla, or P. senega L. Preferably, the root, stem, or leaf ofPolygala or a mixture of them is used in the present invention, morepreferably, the root of Polygala, and most preferably, the cortex of theroot is used.

Preferably, said polar solvent extract i) is prepared by decoctingPolygala with said polar solvent under refluxing for 0.5–10 hours, andrecovering a liquid portion from the decocted mixture by a solid-liquidseparation means as said polar solvent extract, and optionallyconcentrating said liquid portion to obtain a concentrate as said polarsolvent extract.

Preferably, said organic solvent used in the extraction for preparingsaid aqueous fraction ii) is ethyl acetate or butanol.

Preferably, said polar solvent extract i) is used to obtain said organiceluate iii).

Preferably, said aqueous fraction ii) is used to obtain said organiceluate iii).

Preferably, said organic solvent used in said elution is ethanol, or amixture of water and ethanol. More preferably, said organic solvent usedin said elution is a mixture of water and ethanol containing 10–95volume % of ethanol.

Preferably, the pharmaceutical composition comprises said polar solventextract i) as said active ingredient.

Preferably, the pharmaceutical composition comprises said aqueousfraction ii) as said active ingredient.

Preferably, the pharmaceutical composition comprises said organic eluateiii) as said active ingredient.

Preferably, the pharmaceutical composition comprises said filtratehaving a molecular mass less than 30000 Dalton iv) as said activeingredient. More preferably, the pharmaceutical composition comprises afiltrate having a molecular mass less than 3000 Dalton in said filtratehaving a molecular mass less than 30000 Dalton iv) as said activeingredient

The present invention also disclose a method of preparing apharmaceutical composition and the pharmaceutical composition preparedby said method, which comprises the following steps:

-   -   I) extracting Polygala with water to obtain a water extract;    -   II) introducing the water extract from step I) into a reverse        phase chromatography column and eluting the column in sequence        with water and an organic solvent; and    -   III) filtrating the resulting organic elute from step II) with a        molecular sieve to obtain a filtrate having a molecular mass        less than 30000 Dalton, preferably less than 3000 Dalton.

Preferably, said extracting in step I) comprises decocting a mixture ofPolygala with water under refluxing for 0.5–10 hours, and recovering aliquid portion from the decocted mixture by a solid-liquid separationmeans as said water extract, and optionally concentrating said liquidportion to obtain a concentrate as said water extract; and said organicsolvent in step II) is ethanol or a mixture of water and ethanol. Morepreferably, said organic solvent in step II) is a mixture of water andethanol containing 10–95 volume % of ethanol.

A suitable reverse phase chromatography column for use in the method ofthe present invention includes (but not limited thereto) a reverse phasechromatography column packed with a porous resin, for examples DiaionHP-20 (Mitsubishi Co.), Sephadex LH-20 (Pharmicia Co.) and RP-18(Nacalai tesque Co.).

Without further elaboration, it is believed that the above descriptionhas adequately enabled the present invention. The following specificexamples are, therefore, to be construed as merely illustrative, and notlimitations on the remainder of the disclosure in any way whatsoever.

EXAMPLE 1

Dry whole root of P. tenuifolia Willd. was cooked in water in an ratioof 1 g 10 ml under refluxing for one hour. An aqueous extract wasobtained after filtering the cooked mixture with a mesh No. 350 sieve.The aqueous extract was concentrated by evaporation in vacuo, and theresulting concentrate was dried by lyophilization to yield a powderproduct, which is designated as W.

EXAMPLE 2

The procedures in Example 1 were repeated except that a dry cortex ofthe root was used to replace the dry whole root. The powder productyielded in this example is designated as C.

EXAMPLE 3

The aqueous extract from Example 1 was partitioned with an equal volumeof butanol. The resulting organic phase and aqueous phase, after beingseparated, were respectively concentrated in vacuo, and dried bylyophilization to yield two powder products, which are designated asW-BP (the organic partition) and W-BP-H₂O (the aqueous partition).

EXAMPLE 4

The procedures in Example 3 were repeated except that ethyl acetate wasused to replace the butanol. The powder products obtained in thisexample are designated as W-EP (the organic partition) and W-EP-H₂O (theaqueous partition).

EXAMPLE 5

The aqueous extract from Example 2 was partitioned with an equal volumeof ethyl acetate. The resulting aqueous phase was separated from thepartitioned mixture, and partitioned with an equal volume of ethylacetate. The twice-partitioned aqueous phase was separated from themixture, and partitioned with an equal volume of ethyl acetate. Theresulting organic phase and aqueous phase from the third-timepartitioned mixture after being separated, were respectivelyconcentrated in vacuo, and dried by lyophilization to yield two powderproducts, which are designated as C-3EP (the organic partition) andC-3EP-H₂O (the aqueous partition).

EXAMPLE 6

The aqueous extract from Example 2 was concentrated to become asemi-fluid extract, and was subjected to a reverse phase chromatographyin a ratio of 1 g dry power to 20–120 g resin. To a reverse phasechromatography column packed with Diaion HP-20 resin having a diameterof 500 μm-800 μm the concentrated extract was injected. Water and 95%ethanol each having a volume three times of the resin were used insequence to carry out elution. The water eluate and 95% ethanol eluatewere collected separately, concentrated in vacuo, and dried bylyophilization to yield two powder products, which are designated asC-RC95-H₂O (water eluate) and C-RC95 (95% ethanol eluate), respectively.

EXAMPLE 7

The procedures in Example 6 were repeated except that 70% ethanolaqueous solution was used to replace the 95% ethanol in the elution. Thepowder product obtained from the 70% ethanol eluate is designated asC-RC70.

EXAMPLE 8

The procedures in Example 6 were repeated except that 50% ethanolaqueous solution was used to replace the 95% ethanol in the elution. Thepowder product obtained from the 50% ethanol eluate is designated asC-RC50.

EXAMPLE 9

The 70% ethanol eluate from Example 7 was concentrated 20 times andfiltrated with a molecular sieve having a cut at 3000 Dalton (purchasedfrom Millipore Co., code number: S1Y3) to yield a retentate and apermeate having a molecular mass less than 3000 Dalton. The permeate wasconcentrated in vacuo and dried by lyophilization to yield a powerproduct, which is designated as C-RC70-3000.

EXAMPLE 10

The procedures in Example 9 were repeated except that a molecular sievehaving a cut at 1000 Dalton (purchased from Millipore Co., code number:S1Y1) was used to replace the S1Y3 molecular sieve. The powder producthaving a molecular mass less than 1000 Dalton obtained in this exampleis designated as C-RC70-1000.

EXAMPLE 11

The procedures in Example 9 were repeated except that a molecular sievehaving a cut at 10000 Dalton (purchased from Millipore Co., code number:S1Y10) was used to replace the S1Y3 molecular sieve. The powder producthaving a molecular mass less than 10000 Dalton obtained in this exampleis designated as C-RC70-10000.

EXAMPLE 12

The procedures in Example 9 were repeated except that a molecular sievehaving a cut at 30000 Dalton (purchased from Millipore Co., code number:S1Y30) was used to replace the S1Y3 molecular sieve. The powder producthaving a molecular mass less than 30000 Dalton obtained in this exampleis designated as C-RC70-30000.

Evaluation of the Anti-Depression Activity

The anti-depression activity of the extract was evaluated by thetetrabenazine test [Gylys J. et al., Annals NY Acad. Sci., 107: 899,1963; Maryanoff B. E. et al., J. Med. Chem., 27: 1067, 1984; KatsuyamaM. et al., Arch. Intern. Pharmacodyn. Thera. 283: 61, 1986]. Solvent(distilled water, 10 ml/kg) or extract was administered PO to a group of3 ICR derived male or female mice weighing 22±2 grams, 60 minutes beforeinjection of tetrabenazine methane sulfonate (TBZ; 75 mg/kg, IP). Bodytemperature was recorded before solvent or extract administration and at60, 90 and 120 minutes after TBZ intraperitoneally injection (IP).Inhibition of TBZ-induced hypothermic response was calculated asfollows:

${{Inhibition}\mspace{14mu}(\%)} = {\frac{\left\lbrack {\left( {\Delta\mspace{14mu}{of}\mspace{14mu}{solvent}\mspace{14mu}{group}} \right) - \left( {\Delta\mspace{14mu}{of}\mspace{14mu}{extract}\mspace{14mu}{group}} \right)} \right\rbrack}{\left( {\Delta\mspace{14mu}{of}\mspace{14mu}{solvent}\mspace{14mu}{group}} \right)} \times 100\%}$wherein Δ represents decrease in body temperature.

Results of the anti-depression activities of Examples 1–4 evaluated byTBZ-induced hypothermia

Dosage^(a)) Inhibition (%) Samples (g/kg) at 60-90-120 minutes W 4—^(b)) W 2 85-68-84 W 1 55-68-82 W 0.5 50-54-62 W 0.25 18-14-28 W-BP 161-63-54 W-BP 0.5 82-70-70 W-BP-H2O 1 100-98-100 W-BP-H2O 0.5 100-76-65W-BP-H2O 0.25 40-24-32 W-EP 1 0-0-0 W-EP-H2O 0.5 100-83-73 W-EP-H2O 0.2528-32-27 ^(a))Dosage: grams/kg of the powder product administered tomouse ^(b))Part of the mice died

Results of the anti-depression activities of Examples 5–12 evaluated byTBZ-induced hypothermia

Dosage^(a)) Inhibition (%) Samples (g/kg) at 60-90-120 minutes C 4—^(b)) C 2 79-86-99 C 1 100-82-81 C 0.5 24-40-23 C-3EP 4 9-21-16 C-3EP 10-0-0 C-3EP-H2O 4 100-91-93 C-3EP-H2O 1 100-84-97 C-3EP-H2O 0.5 77-52-54C-3EP-H2O 0.25 26-23-29 C-3EP-H2O 0.1 14-13-37 C-RC95-H2O 4 46-60-59C-RC95-H2O 2 0-5-21 C-RC95 4 —^(b)) C-RC95 1 100-100-100 C-RC95 0.5100-100-93 C-RC95 0.25 72-71-78 C-RC70 2 —^(b)) C-RC70 0.5 100-100-96C-RC50 4 100-100-95 C-RC50 1 53-47-46 C-RC50 0.5 33-16-29 C-RC50 0.2516-0-0 C-RC70-3000 2 92-92-100 C-RC70-3000 1 100-100-91 C-RC70-3000 0.577-64-49 C-RC70-1000 1 100-94-83 C-RC70-1000 0.5 62-74-71 C-RC70-10000.25 37-33-47 C-RC70-10000 4 100-100-100 C-RC70-10000 1 76-77-65C-RC70-10000 0.5 57-67-66 C-RC70-10000 0.25 18-27-28 C-RC70-30000 1100-100-100 C-RC70-30000 0.5 100-100-81 C-RC70-30000 0.25 62-41-39^(a))Dosage: grams/kg of the powder product administered to mouse^(b))Part of the mice died

1. An anti-depression pharmaceutical composition comprising atherapeutically effective amount of an active ingredient, in admixturewith a pharmaceutically acceptable carrier or diluent for the activeingredient, in which the active ingredient is an organic eluate preparedby a method comprising the following steps i) to iii) or a filtrateprepared by a method comprising the following steps i) to iv): i)extracting Polygala with a polar solvent, the polar solvent being wateror a mixture of water and methanol or ethanol; ii) extracting the polarsolvent extract with an organic solvent, and obtaining an aqueousfraction from said extraction; iii) introducing the polar solventextract or the aqueous fraction into a reverse phase chromatographycolumn, eluting the column with water and an organic solvent, andcollecting an organic eluate; and iv) filtrating said organic eluatewith a molecular sieve to obtain a filtrate having a molecular mass lessthan 30000 Dalton in the organic eluate.
 2. The pharmaceuticalcomposition as claimed in claim 1, wherein step i) comprises decoctingPolygala with said polar solvent under refluxing for 0.5–10 hours, andrecovering a liquid portion from the decocted mixture by a solid-liquidseparation means as said polar solvent extract, and optionallyconcentrating said liquid portion to obtain a concentrate as said polarsolvent extract.
 3. The pharmaceutical composition as claimed in claim2, wherein said organic solvent used in the extraction of step ii) isethyl acetate or butanol.
 4. The pharmaceutical composition as claimedin claim 2, wherein in step iii) said polar solvent extract is used toobtain said organic eluate.
 5. The pharmaceutical composition as claimedin claim 2, wherein in step iii) said aqueous fraction is used to obtainsaid organic eluate.
 6. The pharmaceutical composition as claimed inclaim 5, wherein said organic solvent used in the extraction of step ii)is ethyl acetate or butanol.
 7. The pharmaceutical composition asclaimed in claim 4, wherein said organic solvent used in said elution ofstep iii) is ethanol, or a mixture of water and ethanol.
 8. Thepharmaceutical composition as claimed in claim 5, wherein said organicsolvent used in said elution of step iii) is ethanol, or a mixture ofwater and ethanol.
 9. The pharmaceutical composition as claimed in claim6, wherein said organic solvent used in said elution of step iii) isethanol, or a mixture of water and ethanol.
 10. The pharmaceuticalcomposition as claimed in claim 7, wherein said organic solvent used insaid elution of step iii) is a mixture of water and ethanol containing10–95 volume % of ethanol.
 11. The pharmaceutical composition as claimedin claim 8, wherein said organic solvent used in said elution of stepiii) is a mixture of water and ethanol containing 10–95 volume % ofethanol.
 12. The pharmaceutical composition as claimed in claim 9,wherein said organic solvent used in said elution of step iii) is amixture of water and ethanol containing 10–95 volume % of ethanol. 13.The pharmaceutical composition as claimed in claim 1, wherein saidactive ingredient is said organic eluate resulting from step iii). 14.The pharmaceutical composition as claimed in claim 2, wherein saidactive ingredient is said organic eluate resulting from step iii). 15.The pharmaceutical composition as claimed in claim 4, wherein saidactive ingredient is said organic eluate resulting from step iii). 16.The pharmaceutical composition as claimed in claim 7, wherein saidactive ingredient is said organic eluate resulting from step iii). 17.The pharmaceutical composition as claimed in claim 8, wherein saidactive ingredient is said organic eluate resulting from step iii). 18.The pharmaceutical composition as claimed in claim 9, wherein saidactive ingredient is said organic eluate resulting from step iii). 19.The pharmaceutical composition as claimed in claim 10, wherein saidactive ingredient is said organic eluate resulting from step iii). 20.The pharmaceutical composition as claimed in claim 11, wherein saidactive ingredient is said organic eluate resulting from step iii). 21.The pharmaceutical composition as claimed in claim 12, wherein saidactive ingredient is said organic eluate resulting from step iii). 22.The pharmaceutical composition as claimed in claim 1, wherein saidactive ingredient is said filtrate having a molecular mass less than30000 Dalton resulting from step iv).
 23. The pharmaceutical compositionas claimed in claim 2, wherein said active ingredient is said filtratehaving a molecular mass less than 30000 Dalton resulting from step iv).24. The pharmaceutical composition as claimed in claim 4, wherein saidactive ingredient is said filtrate having a molecular mass less than30000 Dalton resulting from step iv).
 25. The pharmaceutical compositionas claimed in claim 7, wherein said active ingredient is said filtratehaving a molecular mass less than 30000 Dalton resulting from step iv).26. The pharmaceutical composition as claimed in claim 8, wherein saidactive ingredient is said filtrate having a molecular mass less than30000 Dalton resulting from step iv).
 27. The pharmaceutical compositionas claimed in claim 9, wherein said active ingredient is said filtratehaving a molecular mass less than 30000 Dalton resulting from step iv).28. The pharmaceutical composition as claimed in claim 10, wherein saidactive ingredient is said filtrate having a molecular mass less than30000 Dalton resulting from step iv).
 29. The pharmaceutical compositionas claimed in claim 11, wherein said active ingredient is said filtratehaving a molecular mass less than 30000 Dalton resulting from step iv).30. The pharmaceutical composition as claimed in claim 12, wherein saidactive ingredient is said filtrate having a molecular mass less than3000 Dalton resulting from step iv).
 31. The pharmaceutical compositionas claimed in claim 1, wherein said filtrating in step iv) yields afiltrate having a molecular mass less than 3000 Dalton, and said activeingredient is said filtrate having a molecular mass less than 3000Dalton.
 32. The pharmaceutical composition as claimed in claim 2,wherein said filtrating in step iv) yields a filtrate having a molecularmass less than 3000 Dalton in said filtrate having a molecular mass lessthan 30000 Dalton iv).
 33. The pharmaceutical composition as claimed inclaim 4, wherein said filtrating in step iv) yields a filtrate having amolecular mass less than 3000 Dalton, and said active ingredient is saidfiltrate having a molecular mass less than 3000 Dalton.
 34. Thepharmaceutical composition as claimed in claim 7, wherein saidfiltrating in step iv) yields a filtrate having a molecular mass lessthan 3000 Dalton, and said active ingredient is said filtrate having amolecular mass less than 3000 Dalton.
 35. The pharmaceutical compositionas claimed in claim 8, wherein said filtrating in step iv) yields afiltrate having a molecular mass less than 3000 Dalton, and said activeingredient is said filtrate having a molecular mass less than 3000Dalton.
 36. The pharmaceutical composition as claimed in claim 9,wherein said filtrating in step iv) yields a filtrate having a molecularmass less than 3000 Dalton, and said active ingredient is said filtratehaving a molecular mass less than 3000 Dalton.
 37. The pharmaceuticalcomposition as claimed in claim 10, wherein said filtrating in step iv)yields a filtrate having a molecular mass less than 3000 Dalton, andsaid active ingredient is said filtrate having a molecular mass lessthan 3000 Dalton.
 38. The pharmaceutical composition as claimed in claim11, wherein said filtrating in step iv) yields a filtrate having amolecular mass less than 3000 Dalton, and said active ingredient is saidfiltrate having a molecular mass less than 3000 Dalton.
 39. Thepharmaceutical composition as claimed in claim 12, wherein saidfiltrating in step iv) yields a filtrate having a molecular mass lessthan 3000 Dalton, and said active ingredient is said filtrate having amolecular mass less than 3000 Dalton.